Tomato brown rugose fruit virus: An emerging and rapidly spreading plant RNA virus that threatens tomato production worldwide

Abstract Tomato brown rugose fruit virus (ToBRFV) is an emerging and rapidly spreading RNA virus that infects tomato and pepper, with tomato as the primary host. The virus causes severe crop losses and threatens tomato production worldwide. ToBRFV was discovered in greenhouse tomato plants grown in Jordan in spring 2015 and its first outbreak was traced back to 2014 in Israel. To date, the virus has been reported in at least 35 countries across four continents in the world. ToBRFV is transmitted mainly via contaminated seeds and mechanical contact (such as through standard horticultural practices). Given the global nature of the seed production and distribution chain, and ToBRFV's seed transmissibility, the extent of its spread is probably more severe than has been disclosed. ToBRFV can break down genetic resistance to tobamoviruses conferred by R genes Tm‐1, Tm‐2, and Tm‐2 2 in tomato and L 1 and L 2 alleles in pepper. Currently, no commercial ToBRFV‐resistant tomato cultivars are available. Integrated pest management‐based measures such as rotation, eradication of infected plants, disinfection of seeds, and chemical treatment of contaminated greenhouses have achieved very limited success. The generation and application of attenuated variants may be a fast and effective approach to protect greenhouse tomato against ToBRFV. Long‐term sustainable control will rely on the development of novel genetic resistance and resistant cultivars, which represents the most effective and environment‐friendly strategy for pathogen control. Taxonomy Tomato brown rugose fruit virus belongs to the genus Tobamovirus, in the family Virgaviridae. The genus also includes several economically important viruses such as Tobacco mosaic virus and Tomato mosaic virus. Genome and virion The ToBRFV genome is a single‐stranded, positive‐sense RNA of approximately 6.4 kb, encoding four open reading frames. The viral genomic RNA is encapsidated into virions that are rod‐shaped and about 300 nm long and 18 nm in diameter. Tobamovirus virions are considered extremely stable and can survive in plant debris or on seed surfaces for long periods of time. Disease symptoms Leaves, particularly young leaves, of tomato plants infected by ToBRFV exhibit mild to severe mosaic symptoms with dark green bulges, narrowness, and deformation. The peduncles and calyces often become necrotic and fail to produce fruit. Yellow blotches, brown or black spots, and rugose wrinkles appear on tomato fruits. In pepper plants, ToBRFV infection results in puckering and yellow mottling on leaves with stunted growth of young seedlings and small yellow to brown rugose dots and necrotic blotches on fruits.


| INTRODUC TI ON
Tomato (Solanum lycopersicum) and pepper (Capsicum annuum) are major field and greenhouse vegetable crops grown all over the world (Baenas et al., 2019;Quinet et al., 2019). Like other cultivated crops, tomato and pepper suffer from constant attacks by various pests and pathogens. Viruses are among a few major pathogens that impede tomato and pepper production, and important viruses include, but are not limited to, begomoviruses, tospoviruses, cucumoviruses, potyviruses, and tobamoviruses. Infection by these viruses reduces crop yield and deteriorates fruit quality and marketability, causing substantial economic losses (Hanssen & Lapidot, 2012; Jones & Naidu, 2019; Moury & Verdin, 2012). Historically, the genus  (ICTV, 2021) In addition to these existing viral pathogens, newly emerging viral diseases also pose a serious threat to tomato and pepper production. Recently, Tomato brown rugose fruit virus (ToBRFV), a new virus in the genus Tobamovirus, has been identified from tomato plants (Luria et al., 2017;Salem et al., 2016). The virus has caused devastating disease outbreaks in tomato production areas in many countries, resulting in a severe reduction in yield (Avni et al., 2021;EPPO, 2020;Jones, 2021;Oladokun et al., 2019).
Currently, ToBRFV is considered the most serious threat to tomato production in the world. showed typical viral symptoms (Salem et al., 2016). In this outbreak, the disease incidence was close to 100%. Although foliar symptoms were apparently mild at the end of the season, brown rugose symptoms on fruits were strong, which greatly affected fruit marketability. Subsequent diagnosis of these tomato plants with molecular biology and bioinformatics tools identified the causal agent to be a new tobamovirus and the virus was named Tomato brown rugose fruit virus (Salem et al., 2016). Shortly after this work, the Dombrovsky laboratory in Israel also reported the discovery of a new tobamovirus isolate from tomato plants grown in net houses in southern Israel in October-November of 2014 where the infected plant displayed mild to severe foliar symptoms and yellowing spots on fruits (Luria et al., 2017). A comprehensive molecular and morphological study was carried out to characterize the virus causing the outbreak. The Israeli isolate (GenBank accession no. KX619418) was found to share high sequence identity with the Jordanian isolate (KT383474). Thus, the first outbreak of ToBRFV was traced back to October 2014 in Israel. Moreover, Luria and co-workers also discovered that ToBRFV could infect tomato cultivars carrying Tm-1, Tm-2, or Tm-2 2 and infect pepper cultivars (Luria et al., 2017).

| Discovery and distribution
After the discovery of ToBRFV in Jordan and Israel, the virus seemed to spread rapidly as the list of countries having ToBRFV has expanded very quickly. To date, the number has reached 35 across four continents, including Asia, Europe, North America, and Africa ( Figure 1 and Table 1). As shown in Figure 1, these countries are mainly in the Middle East and Europe. Given the global nature of the seed production and distribution chain and ToBRFV's seed transmissibility, the extent of its spread is believed to be more severe than has been recorded. Consistent with this assumption, although the virus has not yet been officially reported from countries such as Australia, Peru, India, Ethiopia, and Japan, some tomato and pepper seeds exported from these countries to European and North American countries were found to be ToBRFV-contaminated, suggesting that ToBRFV is highly likely to be present in these countries (EPPO, 2020(EPPO, , 2022.

| Transmission
In protected facilities such as greenhouses, ToBRFV is transmitted primarily by mechanical contact, including propagation materials, plant debris, contaminated soil, growing media, circulating water, workers' farming activities, and culture tools (Dombrovsky & Smith, 2017;Oladokun et al., 2019). Plants damaged by both abiotic and biotic factors may be more susceptible to tobamovirus infection

TA B L E 1
List of first reports of ToBRFV across the world (Dorokhov et al., 2018). Interestingly, the emission of methanol resulting from mechanical damage is likely to promote tobamovirus transmission between neighbouring plants (Dorokhov et al., 2012).
Tomato seeds extracted from ToBRFV-infected fruits are 100% contaminated , although the virus is only detected externally on the seed coat (testa) (Klap et al., 2020a;Salem et al., 2022). Nevertheless, like all other seedborne viruses, the seed transmission rate from ToBRFV-contaminated seeds to their seedling is low, ranging from 0.08% to 2.8% Salem et al., 2022). These results suggest that seed transmission may establish some initial infection foci; further spread to cause an outbreak is through various types of mechanical contact.
International seed imports and exports are indispensable for global food security and sustainable agriculture (Dombrovsky & Smith, 2017). However, such international movement makes it possible for the long-distance dissemination of seedborne viruses such as ToBRFV (Rizzo et al., 2021). For instance, several European countries, such as Spain and the Netherlands, have detected and intercepted some ToBRFV-positive seed packages imported to their countries (EPPO, 2021). Furthermore, transportation of damaged ToBRFV-contaminated fruits may also contribute to the longdistance transmission of ToBRFV, albeit intact tomato fruits are not likely to transmit the virus Klap et al., 2020a).
It is generally believed that there are no specific insect vectors that transmit ToBRFV (Oladokun et al., 2019). However, a recent study has shown that the bumblebee (Bombus terrestris), extensively used as a pollinator for tomato production, can transmit ToBRFV from hives carrying infectious inoculum to healthy tomato plants via buzz pollination (Levitzky et al., 2019). Thus, insect activities such as bumblebee pollination may accelerate the spread of the virus and bumblebees could be used to monitor greenhouses for the presence of ToBRFV. Panno and colleagues found that ToBRFV starting from two infected tomato plants could spread to an entire greenhouse, with an incidence rate of nearly 100%, in the presence of two bumblebee hives within a 9-month monitoring period .
As a proper control was lacking, the possible role of bumblebees in ToBRFV spread in this study needs to be confirmed. A more recent spatiotemporal investigation in tomato commercial greenhouses revealed that during a 24-week culture period, ToBRFV was marginally aggregated at the initial stage, but vastly aggregated during the exponential phase of infection (González-Concha et al., 2021). In this case, it was not clear if bumblebees were provided for pollination or not.

| Disease symptoms
Symptoms resulting from ToBRFV infection are very similar to those from other tobamoviruses such as ToMV (Alon et al., 2021). Virusinduced foliar symptoms are more obvious in young leaves at the top of plants. Typical symptoms on tomato include mosaic, chlorotic, mottling, and deformed leaves, and necrotic spotted or brown rugose fruits. Symptom severity may vary among different cultivars, plants, growth stages, and culture conditions ( Figure 2). For instance, fruits of greenhouse tomato plants infected with the Jordanian isolate displayed strong brown rugosity on fruit, while in contrast foliar symptoms were found to be mild (Salem et al., 2016). Infected tomato plants grown in net houses in southern Israel showed mild to severe mosaic symptoms on leaves and 10%-15% of fruits from diseased plants were yellow-spotted (Luria et al., 2017). Foliar symptoms such as leaf narrowing, chlorotic mottling, and dark green bulges were documented in reports from Germany and the Netherlands (Menzel et al., 2019;van de Vossenberg et al., 2020), and drying and brown necrosis patches on the pedicles, calyces, and flowers were observed in the United States and China (Chanda et al., 2021b;Yan et al., 2019). Infection by ToBRV can reduce fruit yield by 15%-55% regardless of whether or not tested tomato cultivars carry the resistance gene Tm-2 2 (Avni et al., 2021).
In  also a major tomato virus that impacts tomato production worldwide . One of the major PepMV control strategies is cross-protection by preinoculation of attenuated mild PepMV strains, which is widely used in greenhouse tomato production.

| Mixed infection
However, a recent study showed that tomato plants coinfected by ToBRFV and a mild PepMV strain CH2 induced severe new viral disease symptoms including open or scarred unripe fruits and various leaf phenotypes such as bubbling, yellow patches, narrowing or serrated margins (Klap et al., 2020b). By sequential inoculations of tomato plants with ToBRFV and the PepMV mild isolate, they found that preinoculation of ToBRFV enhanced PepMV titres and induced symptoms characteristic of PepMV aggressive strains (Klap et al., 2020b). Moreover, when fruits infected by ToBRFV and PepMV were damaged, they could serve as an effective inoculum source (Klap et al., 2020a). These observations raise serious concerns about the application of mild PepMV strains to cross-protect against severe PepMV when ToBRFV is endemic. ToBRFV MP is the key factor for overcoming Tm-2 2 -mediated resistance Yan et al., 2021a). The coat protein (CP) encoded by ORF4 has a predicted mass of 17.5 kDa and is involved in viral particle assembly and long-distance movement (Ishibashi & Ishikawa, 2016). As ELISA is a fast, sensitive, and cost-effective approach for virus detection and diagnosis, efforts have been made to solve the specificity issue due to cross-reaction with other tobamoviruses.

| G ENOME ORG ANIZ ATION AND SEQUENCE DIVERSIT Y
Two monoclonal antibodies that sensitively and specifically recognize ToBRFV CP without serological cross-reactions with TMV and

| MP IS RE S P ON S IB LE FOR OVERCOMING TM-2 2 -MED IATED RE S IS TAN CE
Several R genes, including N, Tm-1, Tm-2, Tm-2 2 , L 1 , L 2 , L 3 , and L 4 , have
To explore the underlying mechanism by which ToBRFV breaks down Tm-2 2 -mediated resistance, Maayan et al. (2018)  ToBRFV and the molecular mechanism underlying these responses.

| Quarantine and phytosanitary measures
Currently, there are no chemicals that can be used to cure ToBRFV- As ToBRFV is a seedborne virus and the introduction of this virus into uninfected areas is believed to occur through contaminated seeds, it is highly recommended to use virus-free tomato and pepper seeds that are harvested from healthy parental plants .
In addition to using healthy seeds, the production site should be ToBRFV-free. As ToBRFV is very stable and highly contagious, crop rotation helps but has limited effect.

| Cross-protection with attenuated variants
Cross-protection is a promising, potent proactive approach for the control of ToBRFV. Essentially, the concept was developed based on the discovery a century ago that preinfection with an attenuated variant of TMV protects tobacco plants against a severe TMV strain (Pechinger et al., 2019;Wagemans et al., 2022;Ziebell & Car, 2010;Ziebell & MacDiarmid, 2017). The possible molecular mechanisms, that is, RNA silencing and exclusion, have been extensively investigated. Cross-protection has been demonstrated to be an effective, practical approach for the control of many plant viruses, including two tobamoviruses (TMV and CGMMV) (Ali et al., 2016;Goto et al., 1984Goto et al., , 1997, Citrus tristeza virus (Costa & Muller, 1980), (Ziebell et al., 2007), Papaya ringspot virus (Yeh & Gonsalves, 1984), PepMV (Chewachong et al., 2015;, and Zucchini yellow mosaic virus (Cho et al., 1992). A representative example is CGMMV. Widespread CGMMV epidemics, enhanced with the difficulty of disinfecting contaminated greenhouses and the additional introduction of the new virus each year via contaminated seed, is a major problem for indoor cucurbit crop production worldwide. An attenuated strain (SH33b) of CGMMV, derived from its parental severe strain SH through ultraviolet irradiation, has been used for the effective control of CGMMV in greenhouse muskmelon in Japan (Motoyoshi & Nishiguchi, 1988). With the availability of powerful molecular biology and bioinformatics tools, the development of attenuated ToBRFV variants in a relatively short time frame is possible. Essentially, one can construct an infectious clone, conduct comparative genome analysis of the ToBRFV genome sequence with other attenuated tobamoviruses to find potential nucleotides of interest, introduce substitution mutations into these, and finally examine their suitability for cross-protection.

| Genetic resistance
Genetic resistance represents the most effective, economical, and sustainable approach in the control of viral diseases, as it is environmentally friendly, target-specific, and provides reliable protection without additional labour or material costs during the growing season (Nicaise, 2014;Wang, 2015). Unfortunately, no ToBRFVresistant cultivars are currently commercially available. Tomato germplasms, particularly its close relatives, are important sources of genetic resistance to viruses. Previously, Tm-1, Tm-2, and the durable tobamovirus-resistant gene Tm-2 2 were all identified from wild tomatoes (Tm-1 from S. habrochaites, Tm-2 and Tm-2 2 from S. peruvianum) (Hall, 1980;Lanfermeijer et al., 2003;Pelham, 1966).
These genes were introgressed into cultivated tomatoes by breeders over several generations. Screening of 636 Solanum accessions from sections Lycopersicon and Juglandifolia resulted in the identification of three S. ochrantum accessions (LA2160, LA2688, and LA1385) highly resistant to ToBRFV (Jewehan et al., 2022). As these three accessions can also restrict TMV and ToMV to inoculation foci, they have great potential as a source of genetic resistance to tobamoviruses for tomato breeding programmes. Another screening of 160 genotypes by Zinger and colleagues identified one resistant and 29 tolerant genotypes . A further inheritance analysis of a selected tolerant genotype and the resistant genotype showed that the tolerance trait is controlled by a single recessive gene whereas the resistance trait is controlled by at least two genes . One more recent screening of 44 tomato materials by Kabas and colleagues identified four accessions, LA1651 (S. pimpinellifolium), LA0716 (S. penellii), LA4117 (S. chilense), and LA2747 (S. chilense), that are tolerant to ToBRFV (Kabas et al., 2022). Although characterization of genetics underlying these identified resistance/tolerance sources and further introgression of the resistance/tolerance genes from wild germplasms into cultivated tomatoes are technically challenging and time-consuming, this work holds great promise to control ToBRFV.
Genetic transformation is a well-established technology that has been extensively used to engineer genetic resistance into elite cultivars against devastating viruses in a relatively short time. Transgenic plants expressing the genes or sequences of a pathogen can provide resistance to the same or related pathogens, which is termed pathogen-derived resistance (PDR). PDR is mediated by RNA silencing (also RNA interference, RNAi), a conserved defence mechanism triggered by double-stranded RNA (dsRNA) in eukaryotes and the primary antiviral response in plants (Guo et al., 2019;Li & Wang, 2019). The Beachy group first demonstrated that transgenic tobacco plants expressing the CP of TMV was partially resistant to TMV (Abel et al., 1986). Antiviral resistance (also the gene silencing effect) was significantly improved by using a transgene expression cassette to generate an intronspliced, inverted repeat RNA sequence, which forms a hairpin RNA or dsRNA on splicing (Smith et al., 2000). resistance to these three viruses (Hameed et al., 2017). In recent years, genetic transformation has also been used for the development of antiviral resistance through transgene-expressed artificial microRNA (amiRNA) targeting specific viruses or, in combination with a CRISPR/Cas system, by the expression of a single-guide RNA targeting a specific virus (Chandrasekaran et al., 2016;Mahas et al., 2019;Miao et al., 2021;Zhang et al., 2018). As microRNA (miRNA) plays a pivotal role in RNAi-mediated defence, it is also possible to engineer genetic resistance through manipulation of tomato-encoded miRNAs to directly target the specific loci of the ToBRFV genome (Gaafar & Ziebell, 2020). In addition, genetic transformation-mediated introduction of R genes is another elegant solution to a number of viruses, including tobamoviruses (Tamborski & Krasileva, 2020). In cases where the R-mediated resistance has been overcome, random mutagenesis as well as stepwise artificial evolution may be employed to generate R mutants to restore resistance (Tamborski & Krasileva, 2020). For instance, Sw-5b confers strong resistance to TSWV and new isolates with two single mutations C118Y or T120N in the NSm protein can overcome Sw-5b-mediated immunity (Huang et al., 2022). Using a stepwise artificial evolution strategy, Huang and colleagues successfully generated two Sw-5b mutants that confer resistance to TSWV in transgenic plants (Huang et al., 2021). All the approaches discussed here can be adapted to engineer genetic resistance to ToBRFV. However, as long as genetic transformation is involved, public concerns about genetically modified organisms still remain as a big hurdle to pass before the commercialization of any possible tomato cultivars with engineered resistance to ToBRFV.
Plant viruses have a small genome with limited coding capacity and thus rely on a variety of host factors, also known as susceptibility factors, to establish successful infection (Wang, 2015). Thus, mutation or silencing of a host factor gene can lead to inheritable recessive genetic resistance to viruses (Hashimoto et al., 2016;Truniger & Aranda, 2009;Wang, 2015). The well-characterized recessive resistance genes eIF4E/4G and their isoform mutants have been validated to be efficient against some plant viruses, particularly potyviruses (Tang et al., 2020;Wang & Krishnaswamy, 2012).
Once host factor genes are identified, the precise genome-editing technology may be employed to mutate or silence them via genetic transformation, followed by removal of the transgene via traditional breeding to obtain transgene-free "green mutants" (Wang, 2015).

TOBAMOVIRUS MULTIPLICATION1 (TOM1) encodes a seven-pass
transmembrane protein that interacts with tobamoviral replication proteins and is indispensable for efficient multiplication of tobamoviruses (Yamanaka et al., 2000(Yamanaka et al., , 2002. Simultaneous mutations of TOM1 and its putative paralog TOM3 restrict tobamovirus infection in Arabidopsis (Yamanaka et al., 2002). Tomato encodes five TOM1 homologous genes. Recently, this group generated tomato quadruple tom1 mutants with CRISPR/Cas9 technology and found that the quadruple-mutant plants grew and developed as wild-type plants but were highly resistant to four tobamoviruses, including ToBRFV (Ishikawa et al., 2022). This exciting work represents a breakthrough in the development of genetic resistance to ToBRFV through advanced biotechnology. An alternative approach for the development of recessive resistance is to generate mutagenized populations induced by chemical and physical mutagens, such as ethylmethane sulfonate (EMS) or gamma-rays, and then screen for target host factor mutants using TILLING or other genomics tools . This approach was successfully used previously to obtain eif4e mutants with resistance to two potyviruses in tomato (Piron et al., 2010). If no host factors are available, one may directly screen mutant populations for novel recessive resistance by infection assay. The availability of powerful high-throughout sequencing and metagenomics tools can accelerate the molecular characterization of the resistance lines identified and the genetics associated with the novel resistance to facilitate breeding for ToBRFV-resistant cultivars.
In addition to the above control strategies, other novel approaches are also under investigation. For example, Iobbi and coworkers reported that autoxidation products of the methanolic extract from Combretum micranthum leaves, 4-hydroxybenzoic acid (the main product of the alkaline autoxidation of the methanolic extract) and catechinic acid (a common product of rearrangement of catechins in a hot alkaline solution), have anti-ToBRFV activity (Iobbi et al., 2022). Molecular docking simulation suggests that 4-hydroxybenzoic acid and catechinic acid target the amino acid residues responsible for ToBRFV CP-CP interactions, which may affect CP structural stability (Iobbi et al., 2022). This work raises the possibility of using natural plant compounds against ToBRFV.

| CON CLUS I ON S AND FUTURE PROS PEC TS
Plant viruses are a major constraint to agriculture, accounting for nearly 50% of newly emerging plant diseases and causing an estimated economic loss greater than $30 billion annually . ToBRFV is a newly discovered, highly contagious, and destructive tobamovirus with tomato as the primary host. Within ToBRFV through cross-protection to provide a fast and effective solution in the short term.
• Screening for resistance in tomato germplasms and subsequent introgression of identified resistance genes into elite cultivars.
Although this approach usually takes a very long time to succeed, positive outcomes of durable resistance will benefit the industry more profoundly.
• Induction of mutagenized tomato populations and screening for novel resistance. The advanced technologies available may accelerate the integration of identified resistance into elite cultivars through breeding programmes.
• New control strategies such as host factor-based resistance. Research The mechanisms underlying these responses are to be investigated.

ACK N OWLED G EM ENTS
The work in the Wang laboratory relevant to this article was supported in part by Agriculture and Agri-Food Canada and a Discovery Grant from the Natural Sciences and Engineering Research Council of Canada (NSERC).

CO N FLI C T O F I NTE R E S T
The authors declare that no competing interests exist.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing is not applicable to this article as no new data were created or analysed.